PROJECT SUMMARY Raspberry ketone [4-(p-hydroxyphenyl)-2-butanone] (RK) is the principal naturally occurring aroma compound from the fruit of red raspberries (Rubus idaeus L.). Currently, RK is marketed and sold in the US as a popular dietary supplement for the management of body weight. One of the primary aims of the parent grant is to examine the bioavailability and metabolic fates of RK in mouse models. The objective of this supplement is to optimize and fully validate our comprehensive analytical methods integrating liquid-liquid phenolic extraction, microelution solid-phase extraction (SPE), lipid removal and sample clean-up, and targeted metabolomics using UHPLC-QqQ- MS/MS, for efficient and reliable analysis of RK and its metabolites in plasma, brain, liver and WAT specimens to support the RK pharmacokinetic and bioavailability studies. For the proposed supplement project, first we will further optimize the method developed for bioanalysis of plasma and expand the application to the lipid-rich tissues e.g. brain, liver and white adipose tissue (WAT) (Aim 1); we will then conduct full validation for the methods developed including the reliability characteristics of Limit of detection (LOD), Limit of quantification (LOQ), Calibration linearity, Compound stability, Accuracy, Repeatability/Precision, Recovery, and Matrix effects following the FDA's Bioanalytical Method Validation Guidance for Industry (Aim 2). Outcomes from this Administrative Supplement studies will ensure sensitive, accurate and reproducible quantitative analysis of RK and derived metabolites following oral administration of RK in obesity prevention and provide innovative and reliable approaches for others to examine RK and RK structurally-related phenolics in RK-enriched botanical preparations in vivo. The proposed project is also relevant to the overall NIH goal of supporting the performance and publication of formal single-laboratory validation studies of quantitative analytical methods.